Richard



y 1960 A. J. PALLOTTA ETAL Re. 24,822

MICROORGANISM CULTURE METHOD AND APPARATUS Original Filed June 22, 1954INVENTOR ARTHUR d PALL07TA DOMLD F 506LWV5K I RIC/MRO Q ThDMAS ATTORNEYSUnited States Patent Ofiice Re. 24,822 Reissued May 3, 1960MJCROORGANISM CULTURE METHOD AND APPARATUS Original No. 2,715,795, datedAugust 23, 1955, Serial No. 438,384, June 22, 1954. Application forreissue March 11, 1959, Serial No. 798,796

25 Claims. CI. 47-58 Matter enclosed in heavy brackets appears in theoriginal patent but forms no part of this reissue specification; matterprinted in italics indicates the additions made by reissue.

This invention relates to methods and apparatus for the propagation ofphotosynthetic microorganisms employing the principle of dialysis.

Among the photosynthetic microorganisms contemplated are algae,including Chlorella, and Chlorella pyrenoidosa are among the more usefulof these microorganisms to which the method and apparatus of the presentinvention have been applied thus far.

The general subject with which the present invention concerns itself israther exhaustively treated in a publication of the Carnegie Instituteof Washington, Publication 600, entitled Algal Culture: From Laboratoryto Pilot Plant, edited by John S. Burlew, published July 15, 1953.According to that publication, the conventional method and apparatus forthe culture of such microorganisms contemplates the addition ofnutrients to an aqueous bath containing the microorganisms as a batchoperation based upon periodic analyses to determine which of thenutrients requires replacementand in what proportions. With suchprocedures, it is very diflicult if nct'impossible to maintainconditions that will assure optimum growth of the microorganismsundergoing the culture.

By way of contrast, in accordance with the present invention, where themicroorganism bath is separated from its nutrient bath by a dialyzingmembrane, replacement of nutrients to the microorganism bath is efiectedon a continuous basis, facilitating the maintenance of the requirednutrients and producing an optimum hydrogen ion concentration for thebest growth conditions. Moreover, by the use of such a dialyzingmembrane, removal of the toxic ingredients from the microorganism bathbecomes automatic, thus again contributing to the attainment of nearlyideal conditions.

Of the commercially available relatively inexpensive materials adaptingitself to the present invention, various forms of cellophane haveproduced excellent results. Such a material, in its regeneratedcellulose form for example, is semi-permeable, excluding bacteria, algaeand other high molecular weight materials yet permitting the passage ofthe nutrient bath ingredients such as those listed on page 94 of theCarnegie Institution of Washington publication already mentioned. Thischaracteristic of semi-permeable or dialyzing membrances permitsordinary sewage to serve as the nutrient bath without endangering thesterility of the medium in which the microorganisms are carried. Anothergreat advantage of the cellophane type of material is its lighttransmitting characteristic permitting the access of natural orartificial light to the microorganism bath for periods of duration andfrequency to produce the optimum growth conditions.

The method of the present invention relates to the propagation ofphotosynthetic microorganisms comprising exposing an aqueous bathcontaining such microorganisms to one surface of a dialyzing membrane,exposing a nutrient bath for the microorganisms to an opposed surface ofthe membrane and exposing the micro organisms to light. It is preferableto agitate the bath containing the microorganisms during the operationbut in any event, movement relative to the membrane is preferablyimparted to one of the baths and eminently satisfactory results havebeen obtained in this connection by imparting the relative movement tothe bath containing the microorganisms and at the same time advancingthis bath along the surface of the membrane.

Highly desirable results have been achieved through the use of themembrane in tubular form and advancing one of the baths therethrough ina substantially helical path.

Under any conditions, the microorganisms in their bath are exposed tolight and such exposure may be intermittent. The introduction of carbondioxide into one or both of the baths has produced excellent resultseven where the introduction of atmospheric air containing only a smallproportion of carbon dioxide is employed. The use of counterfiow for themicroorganism and nutrient baths has indicated further advantages.Although the method of the present invention is applicable to widelydifferent types of photosynthetic microorganisms, its applicability tothe green algae Chlorella pyrenoidosa promises startling results on acommercial scale.

The apparatus contemplated herein comprises a tubular dialyzing membranehaving an inlet and an outlet for the passage of liquid and a receptaclereceiving at least a portion of the membrane for its immersion in asecond liquid. The membrane preferably assumes the form of asubstantially helical tube and agitation means is preferably provided toproduce relative movement between the membrane and receptacle.

A more complete understanding of the invention will follow from a moredetailed description in conjunction with the accompanying drawingwherein:

Fig. 1 is a diagrammatic representation of apparatus adapted for thepracticing of the present invention;

Fig. 2 is a fragmentary View depicting a modified form of tube; and

Fig. 3 is a sectional view taken along line 33 of Fig. 2.

With reference to the drawing, one form of apparatus productive ofhighly satisfactory results includes a tank or receptacle 18 providingjournals 12 rotatably supporting a drum 14 carrying at one of its ends,an internal gear 16 in mesh with a spur gear 18 whose shaft 20 rotatesin a journal 22 also carried by the wall of the tank It), the outer endof the shaft 26 carrying a sprocket 24 driven by, a chain 26 which is inturn driven by a sprocket 28 on the shaft 30 of a motor 32.

Helically wound about the drum or cylinder 14 is a tube 34 having lighttransmitting properties and being semipermeable so as to constitute adialyzing membrane permeable to the nutrient components required for theculture of such microorganisms as those contemplated hereinandimpermeable of course, to the microorganisms themselves. The inlet endof the Cellophane or other dialyzing membrane tube extends through oneof the shafts 36 of the drum, the outlet endpassing through a similarshaft 38, both of which shafts will be provided with rotary joints, notshown, to permit the introduction and discharge of the aqueous bathrelative to the tube without leakage.

Assuming that the microorganism bath. is introduced through the shaft36, tube 34 and shaft 38, then the tank 10 will receive an aqueous bathcontaining the nutrient materials, depicted as having a level 40determined by the adjusted position of an overflow pipe 42 penetrating awall of the tank 10. The nutrient solution can be introduced bydirecting it into the tank by means of a supply pipe 44 and a tube 46connected therewith and/ or through a branch line 48 provided with sprayheads 50.

By the manipulation of valves 52, 54 and 56 arranged in these linesrespectively, the nutrient bath can be introduced through either or bothof the branch lines. Where the spray heads are used, nutrient materialswill be supplied to the dialyzing membranous tube, and therefore themicroorganism bath, even when there is no immersion. It will beunderstood of course, that the two baths can be interchanged so that ifdesired, the nutrient bath could be directed through the dialyzingmembranous tube while the microorganism bath could be introduced to theouter surface thereof.

Rotation of the drum 14 by means of the motor 32 will produce agitationof both the bath within the tube 34 and that within the tank and if thetube is not completely filled with liquid, it will be advanced in thedesired direction through the tube by proper rotation of the drum. Ofcourse, the liquid flowing through the tube can be advanced by the useof differential pressures by means of a pressure head or pumpingapparatus, not shown.

Any number of components such as that shown in Fig. 1 of the drawing canbe connected in series by suitable couplings of conventional types toproduce an optimum length of growing cycle. The rate of rotation of thedrum can assume a value most consistent with the results desired andeven a variable rate can be produced in order to modify the time cycleand/or the light application cycle.

The apparatus depicted in Figs. 2 and 3 contemplates the use of aplurality of cells 6t), 62, 64, 66, 68 and 70 separated one from thenext by means of a dialyzing membrane 72. Assuming that themicroorganism bath is introduced into the cell 60 through an inlet pipe74, it will flow through a connector 76 to the cell 64, through anotherconnector 78 to the cell 68 from which it will be discharged through atubular outlet 80. Similarly, the nutrient bath will be introduced incountercurrent flow into the cell 70 through an inlet tube 82, through aconnector 84 to the cell 66, through a connector 86 to the cell 62 andthrough a discharge tube 88 for disposal or recirculation in a suitablemanner. Where sewage is used as the nutrient bath, it can be processedby the conventional methods and then introduced into an appa ratus ofthe type contemplated herein resulting not only in the utilization ofits nutrient content while excluding any of its contaminants from themicroorganism bath but in oxygenation of the sewage itself which is veryadvantageous in the preservation of marine life.

The light and dark cycles which have been found to be desirable asdescribed in the publication of the Carnegie Institution will occurnaturally where a drum is rotated in the manner described with referenceto Fig. 1 since the microorganisms submerged in the tank carried bathwill be obscured from the light and of course the light itself can beterminated for prescribed periods necessary to achieve the best results.

Temperature conditions can be controlled readily by applying elevated orreduced temperatures to the nutrient bath for example, or by selecting asource of water for such bath as will produce the desired temperatureconditions of the microorganism bath by heat exchange principles.

A most outstanding result achieved in the culture of Chlorellapyrenoidosa by the use of dialysis as described herein is the greatlyincreased yield over previously known methods.

Where natural sunlight is employed, considerable variation will beexperienced. This variation can be cornpensated by modifying the lengthof tubing through which the microorganism bath passes as by changing thenumber of units of the type depicted in Fig. l or 2 to increase ordecrease the length of the path traversed.

At the discharge end of the apparatus, the bath containing themicroorganisms will be withdrawn and the Chlorella pyrenoidosa or otherend products will be separated by means of a centrifuge or otherappropriate methods for use as food, animal feed and other purposes forwhich such end products are suitable.

Whereas the invention has been described with reference to only twospecific forms of apparatus, these examples should not be construed aslimiting nor should the specific aspects of the methods described beconstrued as limiting beyond the scope of the appended claims.

We claim:

l. A method of propagating photosynthetic microorganisms comprisingexposing an aqueous bath containing microorganisms to one surface of adialyzing membrane, exposing a nutrient bath for said microorganisms toan opposed surface of said membrane, and exposing said microorganisms tolight.

2. A method as set forth in claim 1 wherein the bath containing saidmicroorganisms is agitated.

3. A method as set forth in claim 1 wherein movement relative to saidmembrane is imparted to one of said baths.

4. A method as set forth in claim 1 wherein movement relative to saidmembrane is imparted to said microorganism containing bath.

5. A method as set forth in claim 1 wherein said microorganismcontaining bath is advanced along the surface of said membrane.

6. A method as set forth in claim 1 wherein said membrane is tubular andone of said baths flows therethrough.

7. A method as set forth in claim 1 wherein one of said baths isadvanced along a substantially helical path.

8. A method as set forth in claim 1 wherein said microorganisms areexposed to light intermittently.

9. A method as set forth in claim 1 wherein at least one of said bathsis aerated.

10. A method as set forth in claim 1 wherein carbon dioxide isintroduced into at least one of said baths.

11. A method as set forth in claim 1 wherein said baths are advanced inopposite directions relative to said membrane.

12. A method as set forth in claim 1 wherein said microorganisms arealgae.

13. A method as set forth in claim 1 wherein said microorganisms areChlorella.

14. A method as set forth in claim 1 wherein said microorganisms areChlorella pyrenoidosa.

15. A method as set forth in claim 1 wherein said dialyzing membrane isa light transmitting tube serving as a conductor for one of said baths.

16. A method as set forth in claim 1 wherein the diffusion of nutrientsfrom said nutrient bath to said microorganism bath is regulated by saidmembrane to maintain a substantially constant concentration of nutrientsin said microorganism bath.

17. A method as set forth in claim 1 wherein said microorganisms areexposed to light continuously.

18. Apparatus for the propagation of photosynthetic microorganismscomprising a dialyzing membrane in the form of a substantially helicaltube having an inlet and an outlet for the passage of liquidtherethrough, and a receptacle receiving at least a portion of saidmembrane for immersion thereof in a second liquid.

19. Apparatus as set forth in claim 18 wherein said membrane in the formof a substantially helical tube is supported on a rotatable drum.

20. Apparatus as set forth in claim 18 wherein agitation means producesrelative movement between said membrane and receptacle.

21. A method of propagating photosynthetic microorganisms comprising thesteps of: continuously forming a fine dispersion of at least a portionof said culture in the presence of light; exposing said dispersion toone surface of a dialyzing membrane; and exposing a nutrient bath forsaid microorganisms to an opposed surface of said membrane.

22. A method of propagating photosynthetic microorganisms comprising thesteps of: withdrawing at least a portion of an aqueous bath containingmicroorganisms; dispersing said portion in the form of fine particlesinto an atmosphere containing oxygen and carbon dioxide in the presenceof light; returning the light treated particles in the form of finefilms of liquid medium to one surface of a dialyzing membrane; exposinga nutrient bath for said microorganisms to an opposed surface of saidmembrane; and continuously repeating the operation until a desired,predetermined density of algae cells is obtained.

23. The process of claim 22 wherein the forming of the fine dispersionportion of the aqueous bath containing microorganisms is obtained byintermittent spraying of the same.

24. The process of claim 22 which includes the step of continuouslyfeeding and bleeding liquid medium and product respectively to providefor continuous large scale operation.

25. A method of propagating photosynthetic microorganisms comprisingexposing an aqueous bath containing microorganisms to one surface of adialyzing membrane, exposing a nutrient bath for said microorganisms toan opposed surface of said membrane, and exposing said microorganisms toa cycle of higher and lower intensities of light.

References Cited in the file of this patent or the original rpatentUNITED STATES PATENTS Livingston Plant World, vol. 11, No. 2, pp. 39-40(February 1908).

Livington, Plant World, vol. 11, No. 8, pp. 183-184 (August 1908).

